[] Prior Phase II studies with rV-, rF-PSA-TRICOM (PROSTVAC) vaccine as a monotherapy in patients with metastatic castration-resistant prostate cancer (mCRPC) have led to an ongoing Phase III study. This is an ongoing global 3-arm study in patients (n=1,200) with asymptomatic mCRPC who receive (a) PROSTVAC, (b) PROSTVAC + GM-CSF, (c) placebo (empty vector). The endpoint is overall survival (OS). [] We have evaluated the growth rates of tumors in patients with mCRPC receiving PROSTVAC vaccine or several different chemotherapy regimens. These studies have revealed that, unlike chemotherapy, vaccine therapy can reduce the tumor growth rate leading to enhanced survival even in the absence of increases in progression-free survival (PFS). [] We have completed a trial in patients with mCRPC of PROSTVAC vaccination with increasing doses of ipilimumab (anti-CTLA4). No adverse events beyond those seen with ipilimumab alone were observed. These findings provide evidence for the use of vaccine in combination with checkpoint inhibitors such as anti-PDL1. [] Two randomized clinical studies have recently been initiated employing the novel FDA-approved androgen blockade agent enzalutamide +/- PROSTVAC vaccine. The first is in mCRPC patients with a TTP endpoint; the second trial is in non-metastatic patients with rising PSA where changes in PSA velocity at the discontinuation of enzalutamide will be evaluated. [] A trial is in the review process in collaboration with the NCI Cancer Prevention Program. Patients with newly diagnosed low grade prostate cancer will be randomized to active surveillance vs. PROSTVAC vaccination. The endpoints of this study will be a comparison of pre- and post-(1 year) biopsies for tumor extent and immune infiltrate. [] A dual center study in which patients with metastatic breast cancer were randomized to docetaxel +/- PANVAC vaccine (rV-, rF-CEA-MUC1-TRICOM) has recently been completed. We have used a multicolor FACS-based assay to define a peripheral immunoscore monitoring immune cell subsets known in the literature to have specific immune stimulatory or regulatory functions. There was no association of this immunoscore with increased PFS in the docetaxel arm alone (P=0.87), but an association (P0.001) with increased PFS in the vaccine combination arm. [] We have completed a first-in-human Phase I study of recombinant yeast-CEA vaccine in patients with metastatic disease and demonstrated safety to the generation of CEA-specific T-cell responses. This has led to the initiation of a Phase II study in patients with metastatic medullary thyroid cancer (MTC), where the primary endpoint is tumor growth rate following serum calcitonin, which is a marker for disease progression. [] A clinical study has just opened in collaboration with Dr. P. Agarwal in the CCR Urologic Oncology Branch (UOB). Bacillus Calmette-Guerin (BCG) failure bladder cancer patients will be randomized to second-line BCG +/- PANVAC vaccine. The primary endpoint will be tumor extent and immune infiltrate in pre- vs. post-treatment biopsies. [] Numerous immune analyses pre- vs. post-treatment have been/are being employed in clinical studies in an attempt to identify which patients would most likely benefit from a given immunotherapy, and to evaluate potential immune correlates of clinical benefit early in the treatment cycle. It is emphasized that any correlations with clinical outcome using any of the immune assays described must be considered only exploratory. True correlations will require the use of validated assays in larger randomized trials. [] In addition to the use of the Elispot assay to define CD8+ responses in peripheral blood mononuclear cells (PBMCs) to an immunodominant HLA-A2 epitope, we have now developed a FACS-based assay using 15-mer peptides reflecting entire tumor-associated antigen (TAA) gene to simultaneously measure both CD4 and CD8 responses; this assay is not restricted by HLA allele. [] We have developed assays to analyze numerous soluble factors in sera pre- and post-immunotherapy. In collaboration with Dr. J. Gildersleeve in the CCR, we have profiled anti-glycan antibodies using glycan arrays in several of our PROSTVAC trials; levels of Abs to specific glycans significantly (P=0.005) correlated with OS. [] We have analyzed post- vs. pre-serum samples for a range of cytokines as well as two potential immunomodulatory molecules. We have shown that soluble CD27 (sCD27) sera levels are decreased in carcinoma patients vs. healthy donors. mCRPC patients treated with PROSTVAC + ipilimumab showed a significant increase (P0.0005) in sCD27 post-vaccination, which was associated with increased OS (P=0.022). In vitro studies demonstrated that sCD27 provides strong proliferative signals to lymphocytes. [] We have now developed a multi-laser/multi-color FACS-based assay to analyze up to 50 different immune cell subsets in PBMCs pre- vs. post-treatment. In one example, there were associations with OS (P0.005) of several immune cell subsets pre-treatment with PROSTVAC + ipilimumab. [] We have now developed the capability to conduct digital immunohistochemistry (IHC) analyses of biopsy specimens. In a recently completed trial of i.t./s.c. vaccination with PROSTVAC, 19/21 patients developed stable or reduced levels of serum PSA; there were statistical increases in CD4+ and CD8+ T cells, and decreases in Tregs in post- vs. pre-treatment biopsies (P0.001 for each). Immunotherapy trials have been initiated or are planned in prostate, bladder, and lung carcinomas in which pre- vs. post-treatment biopsies will be analyzed by digital IHC and compared with changes in specific immune cell subsets in the periphery. [] We have analyzed the effects of the FOLFOX or FOLFIRI plus bevacizumab regimens on PBMC subsets and have observed no substantial changes in any peripheral immune cell subset. While preliminary, these findings help support the rationale for the use of immunotherapeutics with standard-of-care therapies in patients with metastatic colorectal cancer.